The present invention relates generally to gene therapy for treating chronic heart failure or other cardiac diseases associated with decreased myocardial xcex2-adrenergic receptors and reduced myocardial function. More particularly, this invention relates to manipulation of genes and gene products that affect xcex2-adrenergic receptors in myocardium in order to enhance myocardial function. The invention also relates to receptors, methods or systems for drug screening, and transgenic animals suitable for investigation of therapies for treatment of heart failure and other cardiac conditions.
G-protein-coupled receptors, such as the adrenergic receptors, elevate cellular levels of second messengers like cyclic-adenosine monophosphate (cAMP) and diacylglycerol thereby regulating and coordinating cellular metabolism and function. In the heart, xcex2-adrenergic receptors (xcex2-ARs), responsive to the sympathetic neurotransmitter norepinephrine and the adrenal medullary hormone epinephrine, stimulate adenylyl cyclase, raising myocardial cAMP and increasing cardiac contractility. Elevated circulating catecholamines and myocardial xcex2-AR stimulation represent critical mechanisms for augmenting cardiac function during stress.
As is true for most G protein-coupled receptors, prolonged agonist exposure of xcex2-ARs leads to a rapid decrease in responsiveness. Agonist-dependent desensitization can be initiated by phosphorylation of activated receptors by members of the G protein-coupled receptor kinase (GRK) family (W. P. Hausdorff et al., FASEB J. 4, 2881 (1990); J. Inglese et al., J. Biol. Chem. 268, 23735 (1993)). Phosphorylated receptors then interact with arresting proteins like xcex2-arrestin to which they bind thereby sterically interdicting further coupling to G proteins (W. P. Hausdorff et al., 1990; J. Inglese et al., 1993). The xcex2-adrenergic receptor kinase-1 (xcex2ARK1) is a GRK which has been shown to specifically phosphorylate activated xcex22-ARs in vitro and which is hypothesized to phosphorylate xcex22-ARs in vivo leading to uncoupling and desensitization (W. P. Hausdorff et al., 1990; J. Inglese et al., 1993; M. J. Lohse et al., Proc. Natl. Acad. Sci. U.S.A. 86, 3011 (1989); M. J. Lohse et al., J. Biol. Chem. 265, 3202 (1990); S. Pippig et al., J. Biol. Chem. 268, 3201 (1993)).
The action of xcex2ARK1 on the xcex21-AR has not yet been documented. xcex2ARK1 is specifically targeted to activated receptors in the plasma membrane by a translocation event mediated via a specific protein-protein interaction between the carboxyl terminus of the kinase and the xcex2xcex3 subunits of activated and dissociated G proteins (J. Pitcher et al., Science 257, 1264 (1992); W. J. Koch et al., J. Biol. Chem. 268, 8256 (1993)).
In chronic congestive heart failure, an illness affecting more than four million Americans, there is dramatic impairment of the myocardial xcex2-AR system. Failing human ventricular myocardium contains 50% fewer xcex2-ARs and shows parallel decreases in agonist-stimulated adenylyl cyclase activity and even greater decreases in agonist-mediated inotropy (M. R. Bristow et al., N. Engl. J. Med. 307, 205 (1982), M. R. Bristow et al., J. Mol. Cell. Cardiol. 17 (Suppl. 2), I12 (1990)). In addition, increases in inhibitory G-protein and G-protein receptor kinases (e.g. xcex2-adrenergic receptor kinase) in heart failure may further impair receptor-mediated inotropy (T. Eschenhagen et al., Circulation Research 70, 688 (1992) and M. Ungerer et al., Circulation v7, 454 (1993)). Therapeutic interventions, involving the administration of agonists to stimulate the xcex2-AR/adenylyl cyclase systems have an inherently limited efficacy given the reduction in receptor targets in the diseased myocardium.
An additional possible contributor to the decreased myocardial xcex2-AR responsiveness seen in chronic failing human hearts is that levels of xcex2ARK1 are elevated (M. Ungerer et al., Circulation 87, 454 (1993); M. Ungerer et al., Circ. Res. 74, 206 (1994)). Thus, xcex2-AR impairment in heart failure may have several underlying causes.
The field of transgenic technology has achieved significant advances in techniques for in vivo gene transfer in recent years (T. Ragot et al., Nature 361, 647 (1993); M. A. Rosenfeld et al., Cell 68, 143 (1992); R. J. Guzman, et al., Circulation Research 73, 1202 (1993)).
While several transgenic mice have been reported which express, for example, the c-myc proto-oncogene or SV-40 T-antigen affecting cardiac growth (J. L. Swain et al., Cell 50, 719 (1987); L. J. Field, Science 239, 1029 (1988); E. B. Katz et al., Am. J. Physiol. 262, H1867 (1992)), to date there have been no reports concerning the ability of a transgene to affect myocardial contractility.
The present invention provides novel strategies for improving cardiac function, for example by overexpressing the xcex2-AR in the myocardium, or by inhibiting the activity of xcex2ARK. It utilizes molecular, in vitro and in vivo methodologies to assess the biochemical and physiological consequences of transgenic overexpression of the human xcex22-AR in the heart. In addition, the invention utilizes in vitro and in vivo methodologies to assess the consequences of overexpression or suppression of xcex2ARK in the heart.
In general, the invention features gene therapy for disease states where specific receptor-mediated functions are lost or altered. In particular, defects in the xcex2-adrenergic receptors (B-AR) and in inotropic responsiveness in heart failure are a therapeutic target.
The invention provides a method for the delivery of a gene for xcex2-AR, e.g., xcex22-AR, and a xe2x80x9cminigenexe2x80x9d encoding a xcex2ARK inhibitor for delivery to diseased heart tissue. The invention also provides transgenic mice with cardiac specific overexpression of xcex2-AR or xcex2ARK, and mice with cardiac specific expression of a xcex2ARK inhibitor. In addition, the invention provides a means for screening drugs that may be useful in the treatment of heart disease.
According to the invention, gene transfer to heart tissue may be accomplished by in vivo methods of gene transfer such as those involving the use of recombinant replication deficient adenovirus. Procedures include gene transfer into cardiac muscle are described in the literature, for example in Kass-Eisler, A. et al., Proc. Natl. Acad. Sci. U.S.A., 90: 11498-11502 (1993); Stratford-Perricaudet, L. D. et al., J. Clin. Invest. 90: 626-630 (1992); and Guzman, R. J. et al., Circ. Res. 73: 1201-1207 (1993).
The gene for the human xcex22-AR has been cloned (Kobilka, B. K. et al., Proc. Natl. Acad. Sci. U.S.A. 84:46-50) and in the present invention, the xcex2-AR gene, e.g., xcex22-AR or other subtypes, is simply any nucleic acid sequence which codes for a xcex2-AR, said receptor having the ability to couple to adenylyl cyclase. Thus variations in the actual sequence of the gene can be tolerated provided that the xcex2-AR can be expressed and is able to couple to adenylyl cyclase.
A xcex2-AR gene construct can be obtained through conventional recombinant DNA techniques.
It is an object of the present invention to provide transgenic mice whose germ cells and somatic cells contain overexpressed human xcex2-AR in the heart, transgenic mice whose germ cells and somatic cells contain genes for cardiac overexpression of xcex2ARK, and transgenic mice whose germ cells and somatic cells contain a gene for cardiac expression of a xcex2ARK inhibitor. The transgenic mice of the invention will usually have expressed levels of the gene products in myocardial tissue that are at least 50% greater, and preferably at least 100% greater, than levels than would normally occur in mice. In addition, the transgenic mice""s myocardial function (e.g. heart rate or contractility) can be increased or decreased by about 10% and preferably by about 20% as desired.
Transgenic mice in the present invention were created by microinjection of the desired gene construct into the pronuclei of one cell embryos, which were then surgically re-implanted into pseudo-pregnant female animals. There are several means by which transgenic animals can be made. One method employs the embryonic stem cell methodology known to workers in this field.
Preferably, transcription of the xcex2-AR gene is controlled by a promoter which generates intense cardiac expression. Example of such promoter sequences include, in mice, the xcex1-myosin heavy chain promoter and in larger mammals the CMV (cytomegalo virus) promoter or the xcex2-myosin heavy chain promoter. These examples are not meant to be limiting but rather represent currently available promoters which are felt to be capable of expressing the xcex2-AR at levels above the endogenous, background myocardial xcex2-AR levels.
According to the present invention transgenic mice were created in which the xcex1-myosin heavy chain promoter was utilized to direct intense cardiac specific expression of the human xcex22-adrenergic receptor (ranging from 50 to 200 fold above control levels in three separate transgenic mouse lines). This resulted in dramatic elevation of myocardial adenylyl cyclase activity, enhanced atrial myocardial contractility and increased in vivo left ventricular function; these three parameters at baseline in the transgenic animals, were equal to those observed in control animals maximally stimulated with the xcex2-agonist, isoproterenol.
Accordingly, it is an object of the present invention to provide a recombinant vector for myocardial tissue expression of the xcex2-AR gene.
Preferably, transcription of the xcex2-AR gene coding sequence is controlled by a promoter sequence which generates selective cardiac expression. Examples of such promoter sequences include, in mice, the xcex1-myosin heavy chain promoter and, in humans, the CMV (cytomegalo virus) promoter, or the xcex2-myosin heavy chain promoter. For general use, any promoter which gives large expression or promoter which is only operational in the heart such as those from the genes for xcex1 and xcex2 myosin heavy chain and myosin light chain is suitable. These can be obtained by gene cloning techniques and promoter:fusion gene assays known to those skilled in the art. Typical of these include studies done to characterize promoter regions for the xcex1-myosin heavy chain (Subramian et al., J. Biol. Chem. 266, 24613 (1991) and myosin light chain (Lee et al. J. Biol. Chem., 267, 15875 (1992)).
It is another object of the present invention to provide host cells stably transformed or transfected with the above-described recombinant constructs. The host cell can be prokaryotic (for example, bacterial), lower eukaryotic (for example, yeast or insect), or higher eukaryotic (for example, all mammals, including but not limited to mouse and human). For instance, transient or stable transfections can be accomplished into mammalian myocardial myocytes. Transformation or transfection can be accomplished using protocols and materials known in the art. The transformed or transfected host cells can be used as a source of the recombinant construct.
Transfected cells as described above, such as mammalian myocardial myocytes transformed with a recombinant construct can also be used to treat heart disease by transplanting the transformed myocardial myocytes which express xcex2-AR into a diseased heart tissue. The objective of such. therapy is to increase the heart function of a patient (e.g. strength of contraction) by at least 10%, and preferably by 20% or more.
Thus, it is another object of the invention to provide treatment for heart disease using a myocardial myocyte transformed as described above for transplanting into a diseased heart. Myocytes in culture can be transformed by standard techniques to contain the gene products of the invention. These cells can be delivered to intact myocardium by techniques similar to other muscle grafts and when accepted by the host organ would improve cardiac function by replacing damaged cells with xe2x80x9csuperchargedxe2x80x9d cells.
It is yet another object of the present invention to provide transgenic animals with myocardial overexpression of the human beta-adrenergic receptor. Such transgenic animals can be used as models to study myocardial function and to determine whether increased myocardial xcex2-AR provides resistance to the development of heart failure or ventricular overload. Further, a transgenic mouse according to the present invention can be used to test for agents which decrease heart rate, or block xcex2-AR.
It is yet another object of the invention to provide gene therapy for the treatment of chronic heart failure which is associated with decreased xcex2-ARs.
It is still another object of the invention to provide resistance to heart failure in mammals (experimental animals) by administering the exogenous genes which will increase myocardial xcex2-AR.
It is yet another object of the present invention to provide improved heart function in mammals which exhibit weakened heart function associated with a low number of xcex2-AR receptors, diminished functional activity of the mammals own xcex2-ARs, or decreased contractile function of the left ventricle.
It is a further object of the present invention to provide transgenic animals with myocardial overexpression of xcex2ARK. According to the present invention, transgenic mice were created in which the xcex1-myosin heavy chain promoter was utilized to direct cardiac specific expression of a coding sequence for the entire coding region for bovine xcex2ARK. These mice display marked attenuation of isoproterenol stimulated left ventricular contractility in vivo, and other signs of reduced functioning of the myocardial xcex2-AR system. Such animals have utility for screening potential drugs and therapies to be used for the treatment of heart disease.
Thus, it is a further object of the invention to provide a recombinant vector for myocardial expression of a xcex2ARK inhibitor.
It is yet a further object of the invention to provide a transgenic animal with myocardial expression of a xcex2ARK inhibitor. According to the present invention, transgenic mice were created in which the xcex1-myosin heavy chain promoter was utilized to direct cardiac specific expression of a coding sequence for the carboxyl terminal portion of bovine xcex2ARK which inhibits the activity of the endogenous xcex2ARK. These mice display markedly enhanced cardiac contractility in vivo, even in the absence of a xcex2-agonist.
Therefore, it is yet another object of the invention to provide a recombinant vector for myocardial expression of a xcex2ARK inhibitor. Such vectors can be used therapeutically for conditions where enhancement of functioning in the cardiac xcex2-AR system is desired.
In the present invention, the xcex2ARK gene is any nucleic acid sequence which encodes xcex2ARK, an enzyme that desensitizes xcex2-ARs through a process that involves phosphorylation. A xcex2ARK inhibitor, according to the invention, is any substance that can be delivered to tissue through genetic means that will inhibit the production or functioning of xcex2ARK. A gene for xcex2ARK has been cloned (J. L. Benovic et al., Science 246, 235 (1989)), and xcex2ARK gene construct and xcex2ARK minigene construct can be obtained through conventional recombinant DNA techniques.
The present invention utilizes xcex2ARK1, which is the primary xcex2ARK in the heart. Bovine, mouse, and human xcex2ARK1 have identical functional properties (J. Inglese et al., J. Biol. Chem., 268, 23735 (1993)). The open reading frame of the human xcex2ARK cDNA encodes a protein of 689 amino acids which has 98% amino acid and 92.5% nucleotide identity to bovine xcex2ARK (J. L. Benovic et al., FEBS Lett. 283, 122-126 (1991)). Thus, although bovine xcex2ARK has been used in the examples, the invention includes the claimed vectors, constructs, methods and transgenic animals as made with other xcex2ARK sequences, and in particular with the human sequence.
Preferably, transcription of the ARK gene and the xcex2ARK inhibitor xe2x80x9cminigenexe2x80x9d is controlled by a promoter which generates intense cardiac expression. Examples of such promoter sequences include, in mice, the xcex1-myosin heavy chain promoter and in larger mammals the CMV promoter or the xcex2-myosin heavy chain promoter. Other promoters which result in cardiac-specific expression will also be suitable.
Other features and advantages of the invention will be apparent from the detailed description of the invention, and from the claims. The complete contents of references cited herein are hereby incorporated by reference.